Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Biochimica et Biophysica Acta

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Review

sAC from aquatic organisms as a model to study the evolution ofacid/base sensing☆

Martin TresguerresMarine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA

☆ This article is part of a Special Issue entitled “The RolHealth and Disease,” guest edited by J. Buck and L. R. Levi

E-mail address: mtresguerres@ucsd.edu.

http://dx.doi.org/10.1016/j.bbadis.2014.06.0210925-4439/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: M. Tresguerres, sAActa (2014), http://dx.doi.org/10.1016/j.bba

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 May 2014Accepted 17 June 2014Available online xxxx

Keywords:Cyclic AMPBicarbonateAcidosisAlkalosisCell signalingCarbon Dioxide

Soluble adenylyl cyclase (sAC) is poised to playmultiple physiological roles as an acid/base (A/B) sensor in aquat-ic organisms. Many of these roles are probably similar to those inmammals; a striking example is the evolution-ary conservation of amechanism involving sAC, carbonic anhydrase and vacuolar H+-ATPase that acts as a sensorsystem and regulator of extracellular A/B in shark gills and mammalian epididymis and kidney. Additionally, theaquatic environment presents unique A/B and physiological challenges; therefore, sACs from aquatic organismshave likely evolved distinct kinetic properties aswell as distinct physiological roles. sACs from aquatic organismsoffer an excellent opportunity for studying the evolution of A/B sensing at both the molecular and whole organ-ism levels.Moreover, this information could help understand and predict organismal responses to environmentalstress based on mechanistic models.This article is part of a Special Issue entitled “The Role of Soluble AdenylylCyclase in Health and Disease,” guest edited by J. Buck and L. R. Levin.

© 2014 Elsevier B.V. All rights reserved.

Most aquatic organisms are regularly exposed to fluctuating levels ofCO2, pH and [HCO3

−], both in the environment and in physiologicalfluids, as a result of various conditions such as the balance between pho-tosynthesis and respiration on a day/night cycle, feeding, calcification,upwelling and, in the case of aquatic mammals and birds, diving. More-over, anthropogenic disturbances such as ocean acidification and pollu-tion may induce further acid/base (A/B) disturbances. Because water-breathing animals characteristically have a lower buffering capacity intheir internal fluids compared to air-breathing vertebrates, environ-mental and metabolic disturbances may induce large variations in A/Bhomeostasis that must be constantly sensed and regulated.

Soluble adenylyl cyclase (sAC), which has been identified in aquaticspecies frommultiple phyla (see [1] for a recent compendium), is a goodcandidate as a molecular CO2/pH/HCO3

− sensor of A/B stress in aquaticorganisms. sAC produces the ubiquitous secondmessenger cAMP in re-sponse to elevated CO2/pH/HCO3

− and can potentially mediate multiplephysiological responses via PKA-dependent phosphorylation, ion chan-nel gating and exchange protein activated by cAMP (EPAC) signaling(reviewed in [1–5]). A recent paper has reviewed the established func-tions of sAC in aquatic animals and identified some potential additionalphysiological roles [1]. The current paper further discusses environmen-tal and metabolic conditions that may be related to sAC function in

e of Soluble Adenylyl Cyclase inn.

C from aquatic organisms as adis.2014.06.021

aquatic organisms. It will also discuss the apparent evolutionarily con-servation of sAC-dependent mechanisms in analogous A/B regulatoryorgans from diverse animals and extend the debate on potential func-tions of sAC to phytoplankton.

1. “Aquatic” sAC as a model to study the molecular basis ofA/B sensing

Froman experimental andmethodological perspective, aquatic animalsmay serve as good model systems to study A/B sensing for two main rea-sons. First, they experience large A/B disturbances as part of their normalphysiology. This is a great experimental advantageas it is possible toexposecells and organisms to a wide suite of A/B conditions ranging from normal([HCO3

−]=~5mM;pH~7.8) to alkaline ([HCO3−]=~20mM;pH~8.2) and

to acidic (HCO3−-free; pH ~7.5). The extreme differences between

conditions maximize the magnitude of the responses and readouts,thus facilitating detection and quantification. Most importantly,these extreme conditions are physiologically relevant (compared,for example, to mammalian cells, which are often exposed tonon-physiological HCO3

−-free conditions to maximize responses).Because A/B stress is universal and sAC is evolutionarily conserved,many of these results may also be applicable to other organisms,including humans.

The second reason why sACs from aquatic organisms are interestingmodels is for comparative studies on structure and function. For sAC tobe an evolutionarily conserved A/B sensor as proposed [6,7], its kinetic

model to study the evolution of acid/base sensing, Biochim. Biophys.

2 M. Tresguerres / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

characteristics should vary according to the distinct A/B challenges thateach organismhas regularly experienced throughout their evolutionari-ly history. Because the kinetic differences should be due to key aminoacid residues, mutagenesis studies based on amino acids that differfrom species to species could inform about the molecular basis forsAC's differential sensitivity to HCO3

− and other enzyme kinetic param-eters. For example, the EC50 of dogfish sAC for HCO3

− is ~5 mM [7],which is much lower than the ~20 mM reported for mammalian sAC[6,8,9]. In human sAC, HCO3

− binds between lysine 95 and arginine176, and mutagenesis of the lysine for an alanine completely abolishedthe stimulatory effect of HCO3

− [10]. Alternatively, the sAC amino acidsequences from coelacanth, gar, salmon, trout, chimera and dogfishshark posses an asparagine in a position equivalent to human sAC's ly-sine 95 (Fig. 1).Wouldmutating lysine 95 for asparagine increase the af-finity of human sAC for HCO3

−? Conversely, would mutating theasparagine to lysine reduce the affinity of shark sAC for HCO3

−?Similarly, a comparison between sAC sequences from diverse organ-

isms inform us about potential regulatory mechanisms and interactionswith other proteins. The linker region between catalytic domains 1 and2 in human sAC is 69 amino acids long, has a high proline content andhydrophobicity and has been identified as a potential region for pro-tein–protein interactions (including other sAC domains) [10]. Thelength of the linker region is essentially the same in sAC from all othervertebrates. However, the equivalent region is 148 amino acids long insea urchin sAC and 153 amino acids long in oyster sAC. Do sACs fromthese two invertebrates have additional regulatory properties orinteracting partners? This hypothesis is preliminarily supported bystudies on sea urchin sAC showing co-immunoprecipitation with atleast 10 other proteins [11]. It should also be noted that sea urchinsAC has several PKA phosphorylation sites [12] whereas human sAChas none.

2. Variability of CO2, pH and HCO3− in aquatic environments

Studies to elucidate potential physiological roles of sAC in aquatic or-ganisms require first considering the biology of each organism in rela-tion to its environment. Although the open ocean is chemically stable,some aquatic environments experience pronounced daily or seasonalfluctuations in A/B conditions as a result of biological activity, upwelling,tides or CO2 vents [13]. For example, in coral reefs and kelp forests, thebalance between photosynthesis and respiration can result in daily

Fig. 1.Alignment of sACprotein sequences from diverse animals in the region correspond-ing to amino acids 87–106 of human sAC. Residues in red are conserved across all se-quences, residues in blue are typical of sACs from fish. Arrowheads indicate two keyamino of mammalian sAC: lysine 95 (K) directly binds HCO3

− and aspartate 99(D) coordinates Mg2+ [10]. While D99 is conserved in all sACs, K95 is an asparagine insACs from all fish species. This substitution may help explain the lower EC50 of sharksAC for HCO3

− (~5 mM [7]) compared to mammalian sAC (~20 mM [6,8,9]).

Please cite this article as: M. Tresguerres, sAC from aquatic organisms as aActa (2014), http://dx.doi.org/10.1016/j.bbadis.2014.06.021

shifts of ~0.3 pH units [13,14]. In California kelp forests, even larger var-iations can occur due to sporadic upwelling events of CO2- and nutrient-richwaters that can last for several days [14]. Similar conditionsmay befound in other environments with intense biological activity such asmangroves, algal blooms or oxygen minimum zones. Since in mostcases the changes in pH are due to CO2 production, these are associatedwith equivalent changes in CO2 and HCO3

− following the equilibriumCO2⇔H+ +HCO3

−. Hypercapnia can affect the A/B status in the inter-nal fluids of aquatic organisms by inducing an accumulation of CO2 ininternal fluids due to the reduced gradient for metabolic CO2 excretion(reviewed in [15]), and, in extreme acidic cases, by directly impairingH+ secretion by Na+/H+ exchangers.

While motile animals have the option of moving, they may prefer tostay for food, protection, mating or socializing purposes. Sessile animalsand plants, on the other hand, have no option but to cope with the A/Bstressing conditions. Organisms that do not move must either regulatethe A/B status of their internal fluids or adjust their physiology tofluctu-ating A/B conditions. Thus, the ability to sense A/B must be an adaptiveand widespread trait that undoubtedly regulates multiple physiologicalfunctions in aquatic organisms.

3. Regulation of A/B in extracellular fluids in aquatic animals

An important distinction between the A/B physiologies of aerobicwater- and air-breathing large animals is that the former typicallyhave lower pCO2 and [HCO3

−] in their internal fluids. This is due to thehigh ventilatory requirement for O2 uptake and the high capacitanceof water for CO2 relative to O2 [16]. Since the lower CO2/HCO3

− in inter-nal fluids is associated with a lower buffering capacity, water-breathinganimals experience more pronounced A/B disturbances compared toair-breathers. Furthermore, the changes are proportionally even largerdue the lower CO2, [H+] and [HCO3

−] baseline levels. An additional char-acteristic of aquatic animals with high metabolic demands is that theymust ventilate their respiratory surfaces at a high rate even during rest-ing conditions to be able to extract sufficient oxygen from the surround-ing water. As a result, aquatic animals cannot regulate blood A/B statusby adjusting the ventilation rate (hyper- or hypo-ventilation) like air-breathing animals do. Instead, they rely heavily on metabolic compen-sation, typically by the gills, which act as an analogous organ to mam-malian kidneys (reviewed in [17,18]).

As an example, the post-feeding period is associatedwith ametabol-ic alkalosis both inmammals [19] and sharks [20] as a result of increasedH+ secretion into the stomach and HCO3

− absorption into the blood.However, while in mammals the alkalosis is mild, localized and tran-sient, in sharks it can more than double resting plasma [HCO3

−] (from~ 4 mM to N10 mM, depending on the meal size), elevate pH by ~0.3pHunits, and last ~24h [20–22]. Another example is exhaustive exercis-ing, which in sharks induces severe metabolic acidosis that can almostcompletely deplete plasma [HCO3

−], reduce blood pH by ~0.4 pH unitsand take 4 h to be compensated [23]. A third and final example is thecompensatory A/B response to environmental hypercapnia, which in-volves the accumulation of HCO3

− in plasma to levels that can triple orquadruple baseline levels [15,24,25]. Shark sAC's EC50 for HCO3

− is~5 mM, and its Vmax is ~15mM [7], viz. they respectively match normaland upper plasma [HCO3

−] values found during biologically relevantconditions. Thus, sAC is well suited to be a physiologically relevant ABsensor in sharks ([7], reviewed in [1].

Of the conditions listed above, sAC has to date only been studied inrelation to sensing and counteracting blood alkalosis in shark, towhich it seems essential [7]. Additionally, sAC has been shown to regu-late NaCl and water absorption across the intestine of marine bony fish[26,27]. Promising avenues of future research include regulation of car-diac physiology, vasodilation and vasoconstriction, regulation and coor-dination of aerobic and anaerobic metabolism and regulation of geneexpression. Furthermore, sAC is present in shark red blood cells [1],

model to study the evolution of acid/base sensing, Biochim. Biophys.

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where it may additionally regulate Cl−/HCO3− and Na+/H+ transport in

relation to gas exchange.

4. sAC as a regulator of transepithelial transport

Specialized epithelia such as kidneys and choroid plexus in verte-brates, and gills in fish andmany invertebrates, maintain A/B homeosta-sis in their extra-cellular fluids by secreting and absorbing H+ andHCO3

−: The first report of sAC acting as an epithelial A/B sensor and reg-ulator of H+ transport was in rat epididymis [28] (Fig. 2). The luminalfluid in the distal region of the epididymis is acidic (pH ~6.8) and haslow [HCO3

−] (~5–7 mM) [29], which keep the stored sperm quiescentuntil ejaculation. In response to elevations in luminal pH and [HCO3

−],sAC in “clear” cells becomes stimulated to produce cAMP, leading tothe insertion of more V-type H+-ATPase (VHA) into the apical mem-brane of clear cells, upregulation of H+ secretion and restoration of anacidic luminal fluid [28]. This mechanism also depends on functionalcarbonic anhydrase [28]. Subsequent research established the involve-ment of the actin cytoskeleton [30,31], Ca2+ [30] and PKA [32] in VHAinsertion into the apical membrane, and that the VHA A subunit be-comes phosphorylated [33]. VHA insertion into the apical membraneis negatively modulated by AMPK [33] (reviewed in [34,35]).

Themammalian epididymis is an excellentmodel to study renal acidsecretion because it is easier to isolate andmanipulate compared to kid-ney nephrons, and because the epididymal “clear” cells resemble renalA-intercalated cells (A-IC) both embryologically [36] andmechanistical-ly [34,37]. Indeed, all the sAC-dependent events described above forepididymis also apply to renal A-IC. Both sAC and VHA are abundantlyexpressed in A-ICs [28,38]; VHA insertion into the apical membrane isCA-, sAC-, cAMP- andPKA-dependent [39], and it is negativelymodulated

Fig. 2. Model for acid/base sensing and H+ secretion in mammalian epididymal clear cells acotransporters (NBC) and/or (2) blood CO2 entering via basolateral aquaporins (AQP) stimulainto H+ and HCO3

−. (4) sAC produces cAMP that activates protein kinase A (PKA), which prommembrane for upregulation of acid secretion (5). Potential PKA targets include Ser-175 in theabsorb HCO3

− into the blood via basolateral NBCs and anion exchanger 1 (AE1).

Please cite this article as: M. Tresguerres, sAC from aquatic organisms as aActa (2014), http://dx.doi.org/10.1016/j.bbadis.2014.06.021

by AMPK [39,40]. Studies on kidney have additionally revealed a physicalinteraction between sAC and VHA (based on co-immunoprecipitationand co-immunolocalization) [38], and that the A-subunit becomes phos-phorylated at Ser-175 by PKA [41] and at Ser-384 by AMPK [40]. sAC-,cAMP- and PKA-dependent VHA insertion to the apical membrane alsotakes place in a human salivary gland cell line (HSG) [42]. While cAMPand PKA have the same effect in proximal tubule cells [43], it is yet notknown if sAC is involved.

sAC is also present in renal B-ICs, where it exhibits a bipolar distribu-tion: it colocalizes with VHA in the apical and basolateral region andwith the anion exchanger pendrin in the apical membrane [38]. Al-though this localization suggests a role in regulating HCO3

− secretionand H+ absorption, performing these types of studies is complicatedby the low abundance of B-ICs in relation to A-ICs and principal cells. Al-ternatively, shark gills contain numerous base-secreting cells that areanalogous to renal B-ICs from mammals as they co-express VHA,pendrin [44–46], carbonic anhydrase [47] and sAC [7] and are involvedin HCO3

− secretion and H+ absorption to compensate blood alkalosis [7,47–49]. Furthermore, acid-secreting cells in shark gills do not expressnoticeable amounts of VHA (see references above), and sharks regularlyexperience a pronounced post-feeding alkalosis that is compensatedover several hours (“alkaline tide”) [20]. Furthermore, since sharksonly use H+ and HCO3

− movement to regulate blood pH (they cannotefficiently use ventilatory adjustments, see Regulation of A/B in Ex-tracellular Fluids in Aquatic Animals section), experiments withlive animals are more easily performed and the results more clearlyinterpreted. These characteristics make shark an excellent modelfor studies on the molecular and cellular mechanisms of blood A/Bsensing and regulation, especially those related to HCO3

− secretionand H+ absorption.

nd renal A-intercalated cells (A-ICs). (1) Luminal HCO3− entering via apical Na+/HCO3

−

te sAC (3), in the latter case after cytoplasmic carbonic anhydrase II (CAII) hydrates CO2

otes the insertion of vesicles containing V-type ATPases (VHA, blue icon) into the apicalVHA A subunit, and proteins of the actin cytoskeleton. (6) These acid-secreting cells also

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4 M. Tresguerres / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

The elucidation of the role of sAC in sensing and compensation forblood alkalosis involved experiments with live sharks made alkaloticby intravenous infusion of NaHCO3 combined with pharmacological in-hibitors of CA, microtubule polymerization, and sAC [7,47–49], as wellas sharks in their naturally occurring post feeding period [46,47]. At dif-ferent times after the onset of alkalosis, gill samples were taken and theintracellular localization of VHA determined by immunohistochemistryand, in some cases, byWestern blotting. The resulting cellular model forA/B sensing and regulation (Fig. 3) is mostly amirror image of mamma-lian A-ICs (Fig. 2). Interestingly, post-feeding blood alkalosis results insAC-dependent translocation of VHA from cytoplasmic vesicles tobasolateralmembranes [7], but also in the translocation of the anion ex-changer pendrin to the apical membrane [46]. However, in the lattercase, it is not yet known if sAC is involved. To characterize this modelin more detail (for example, the potential roles of PKA, EPAC andtmACs), it is essential to first develop primary cultures of shark gillcells enriched for base-secreting cells.

Other epithelia also secrete H+ or HCO3− but for physiological func-

tions other than systemic A/B regulation. As examples, the stomach se-cretes H+ for food digestion, the pancreas secretes HCO3

− that activatesdigestive enzymes and neutralizes the acid chyme entering the duode-num, the ciliary body secretes HCO3

− that driveswater transport into theaqueous humor in the eye and the coral calicoblastic epithelium andthe mollusk mantle secrete base to promote calcification. Furthermore,CO2/pH/HCO3

− are known to modulate the transport of other moleculessuch as NaCl andwater acrossmany epithelia. Inmany cases, the effectsof CO2/pH/HCO3

− on transepithelial transport may be explained bythe substrate-law of mass action, by allosteric modulation of iontransporting proteins or by inducing changes in protein structure.However, a detailed examination of the literature also reveals a re-current link between epithelial ion transport and CO2/pH/HCO3

−,carbonic anhydrase and cAMP, three variables intrinsic to sAC

Fig. 3.Model for acid/base sensing andHCO3− secretion in shark gill base secreting cells. (1) Bloo

possibly via aquaporins (AQP). (2) Cytoplasmic carbonic anhydrase II (CAII) hydrates CO2 intotriggers the microtubule-dependent translocation of VHA (blue icon) containing cytoplasmBasolateral VHA reabsorbs H+ into the blood to counteract the original alkalosis. (5) The anionto seawater. sAC is presumed to trigger the translocation of pendrin; however, this has not bee

Please cite this article as: M. Tresguerres, sAC from aquatic organisms as aActa (2014), http://dx.doi.org/10.1016/j.bbadis.2014.06.021

activity. Examples of mammalian epithelia where this link hasbeen described include intestine [50], colon [51], jejenum [52], cho-roid plexus [53] and cornea [54], all of which express sAC [6,55–58].Regulation of epithelial ion transport by CO2/pH/HCO3

− also occursin aquatic animals, for example in crab gills [59], frog stomach [60]and fish intestine [27,61,62] (in the latter example, sAC has alreadybeen shown to be involved [26,27]). Put together, it is likely that sACis a widespread mediator of the effects of CO2/pH/HCO3

− on epithelialion transport by sensing A/B parameters and regulating and coordinat-ing activities of various transporter proteins via cAMP-dependent post-translational modifications. Moreover, apical and basolateral epithelialmembranes may display differential permeabilities to CO2, H+ andHCO3

−, depending on lipid composition and expression of aquaporins,ion channels and transporters [63–65]. This implies that sAC can re-spond to (and sense) the A/B status from one epithelial side and notfrom the other (as shown in isolated dogfish gills in which dfsAC-dependent VHA translocation occurs in response to alkalosis in thebasolateral side but not in the apical side [7]). Finally, sACmay also reg-ulate cell physiology in response to A/B disturbances induced by cellularrespiration in the cytoplasm (e.g., [27,66] and inside mitochondria [67,68].

5. sAC in photosynthesizing aquatic organisms

Marine phytoplankton is composed of many species of diversephotosynthesizing microorganisms that generate half our planet's oxy-gen [69] and are essential to marine foodwebs and the global carboncycle [70]. Some of these microorganisms additionally establish symbi-otic associations with cnidarians, mollusks and other marine animals.Prominent examples are the scleractinian corals, a symbiosis betweencnidarians and dinoflagellates that forms coral reefs, one of the mostbiodiverse ecosystems in the world.

dHCO3− is dehydrated by extracellular carbonic anhydrase IV (CAIV), and CO2 enters the cell

H+ and HCO3−. The elevated intracellular HCO3

− stimulates sAC to generate cAMP, whichic vesicles to the basolateral membrane (4). The targets of cAMP action are unknown.exhanger pendrin (Pd) translocates to the apical membrane to secrete the excess HCO3

−

n experimentally confirmed.

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For photosynthetic organisms, CO2 (alongwith light andwater) rep-resents their food. Photosynthetic activity involves the transport of CO2

and HCO3− and its accumulation in intracellular compartments via car-

bon concentrating mechanisms (CCM). This is essential for photosyn-thesis because the affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (commonly known as RuBisCo) for CO2 is generally muchlower than environmental CO2 levels [71]. Photosynthetic activity isalso associated with substantial changes in pH in the cytoplasm ofaquatic cyanobacteria (~7.2 in the light and ~6.7 in the dark [72]), aswell as in the cytoplasm of coral gastrodermal cells hosting symbioticalgae (~pH 7.4 in the light and ~7.1 in the dark in [73]). Thus, CO2/pH/HCO3

− sensors potentially have several essential roles in coordinatingcell physiology in photosynthesizing organisms.

BLAST searches of genomic and transcriptomic databases revealgenes encoding for putative sAC-like proteins in representatives ofthe major marine phytoplanktonic groups: the diatoms Thalassiosirapseudonana and Phaeodactylum tricornutum [74], cyanobacteria ofthe genus Synechococcus, the foraminifer Emiliania huxleyi and thedinoflagellate Karenia brevis, as well as in corals [75]. The physiologicalroles of sAC in phytoplankton remain unknown; however, experimentswith the diatoms P. tricornutum suggest sAC is involved in regulatinggene expression as cAMP represses the expression of certain genesunder elevated environmental CO2 conditions [76]. Furthermore,P. tricornutum has three adenylyl cyclases that show reasonable conser-vation of amino acids that are key for HCO3

− stimulation in mammaliansAC (e.g., K95), and P. tricornutum lysates demonstrate stimulation ofcAMP by HCO3

− [74]. Similarly, lysates of the diatom T. pseudonanashow HCO3

−-stimulated cAMP production that is sensitive to the sACinhibitor KH7 (Fig. 4).

6. Conclusions and perspectives

Themost fascinating aspects of sAC are that it provides amechanismto sense and respond to a fundamental variable in biology such as A/Bconditions, that sAC and sAC-related enzymes are evolutionarily con-served from cyanobacteria tomammals and that sAC produces the ubiq-uitous signaling molecule cAMP, which can regulate virtually everyaspect of cell biology and physiology. However, for obvious funding rea-sons, most of our understanding on sAC comes from biomedical studies,which, although certainly important, usually lack an evolutionary focus.Comparative studies onmarine organisms have the potential to provideinsights about the evolution of A/B sensingwhile also being relevant forunderstanding and predicting responses to environmental stress.Furthermore, some marine organisms may provide useful models to

Fig. 4. Evidence for sAC-like enzymes in the diatom Thalassiosira pseudonana. (A) RT-PCR of thedatabase annotation. B = blank. (B, C) Stimulation of cAMP production in T. pseudonana extraaliquots were incubated in 100 mM Tris pH 7.5, 5 mM MnCl2, 2.5 mM ATP, 0.5 mM IBMX, 2NaHCO3 = 40 mM. KH7 = 100 μM (control = DMSO) (N = 3).

Please cite this article as: M. Tresguerres, sAC from aquatic organisms as aActa (2014), http://dx.doi.org/10.1016/j.bbadis.2014.06.021

biomedical problems related to A/B sensing and regulation (e.g., sharkbase-secreting cells).

Although sAC ortholog genes are present in a large variety of aquaticanimals [1], it is still for the most part unknown in which cell types sACis expressed, if sAC protein is present inside organelles like shown for afew mammalian cell lines and tissues and what physiological roles sACplays in addition to blood A/B regulation in shark gills [7], NaCl andwater absorption in fish intestines [26,27], and flagellar movementand the acrosome reaction in sea urchin sperm [12,77]. The dual linkof sAC with A/B and cAMP makes the possibilities for additional func-tions endless; however, characterizing sAC function in marine organ-isms entails multiple challenges. To begin with, there is a lack ofexperimental models suitable for reverse genetics, so experiments relyon pharmacological inhibitors such as KH7 and derivatives of catecholestrogens (dCE). Most of our current knowledge on physiology andcell biology has been obtained using this approach, so it is certainlyvery useful. However, this approach complicates studies on live animalsdue to the potential inhibition of sAC in multiple parts of the body.Additionally, the effectiveness of KH7 and dCEs on sAC from each organ-ism, aswell as their specificity for sAC over tmACs, should be confirmed.

This illuminates a second challenge, which is to discern the cellularfunctions regulated by sAC from those regulated by tmACs. The cAMPsignaling microdomain model proposes that cAMP is produced by sACand tmACs in discreet intracellular areas and that cAMP diffusion is re-stricted by phosphodiesterases that degrade cAMP produced in eachmicrodomain, thus preventing (or regulating?) cross-talk and allowingfor signal specificity (reviewed in [2,3]). There is evidence for cAMP-micordomains in marine fish intestine, as sAC stimulates NaCl andwater absorption but tmAC decreases it (and maybe stimulates NaCland water secretion) [26,27]. Furthermore, experiments using forskolinto stimulate tmACs and study their function should be interpreted withcaution because forskolin stimulation of tmACs is so high that is non-physiologically relevant and almost certainly disrupts cAMP micro-domains. The bottom line is that elucidating the roles of sAC requiresmultiple experimental approaches, and that previous results aboutthe roles of cAMP may require a fresh interpretation that considersthe cellular microdomain model.

While there is solid genetic and functional evidence for sAC inelasmobranchs, an open question in fish physiology is if sAC is pres-ent in any other fishes. Although studies on toadfish [27] and seabream [26] intestine suggest sAC is present, those studies relied onWestern blotting with heterologous antibodies against shark sAC,and on pharmacological inhibition with KH7 and dCE. Thus, theycannot be considered definitive evidence. The lack of sAC genes inthe available genomic and transcriptomic databases from zebrafish,

region encoding the catalytic domains of sACs. PID= protein id according to the THASP_3cts by HCO3

−, and sensitivity to KH7, an inhibitor of eukaryote sACs. Diatoms supernatant0 mM creatine phosphate and 100 U.ml−1 creatine phoshpkinase (30 min, 25°C). NaCl,

model to study the evolution of acid/base sensing, Biochim. Biophys.

6 M. Tresguerres / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

fugu, stickleback, pufferfish, killifish and Atlantic cod would seem tosuggest that sAC genes have been lost in some fish lineages. Howev-er, sAC orthologs are definitely present in trout, salmon, gar and coe-lacanth [1], and there is at least one sAC-like gene in catfish. Thisraises technical questions of potential problems in the annotationof some of the fish genomes, but also scientific questions about theevolution of A/B sensing. Why would sAC be present in some fishand in most land vertebrates, but absent in another subset of fishspecies? If this is indeed the case, what genes (if any) have takenover sAC's regulatory roles?

Another question is whether sAC genes from organisms other thanmammals undergo alterative splicing, and if, unlike human, the ge-nomes of some speciesmay containmore than one sAC gene. For exam-ple, there is evidence for more than one sAC gene in corals [75], whichcould have implications for specialization of function of the potentialsAC isoforms based on differential kinetic properties, localizationwithinthe cell and interaction with other proteins.

Finally, it is still unknown whether sAC is present inside organellessuch as mitochondria and nucleus of any organism other than humansand rodents. Unpublished results suggests that sAC is present in thenucleus of cells from coral (Barott and Tresguerres) and shark (Roaand Tresguerres), which could provide a mechanistic model for the reg-ulation of gene expression in response to environmental and metabolicA/B stress based on sAC-dependent phosphorylation of transcription x

Acknowledgements

This study was supported by an Alfred P. Sloan Research Fellowship(grant no. BR2013-103) and a grant from the USA National ScienceFoundation (#EF-1220641). Thanks to Dr. Katie Barott, Dr. VictorVacquier and Dr. Carlos Luquet for their feedback on the manuscript,and to Mr. Jason Ho for bibliography research on the section onphytoplankton.

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